Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells
Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells
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DOI：
10.1002/humu.22041
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发表时间：
2012-04-01
期刊：
影响因子：
3.9
通讯作者：
Lang, Thomas
中科院分区：
文献类型：
--
作者：
Arlanov, Rudolf;Porter, Andrew;Lang, Thomas
多药耐药相关蛋白2(MRP2/ABCC2)参与内源和异种底物的外流，包括几种抗癌和抗病毒药物。ABCC2蛋白变异体的功能结果仍然不一致，这可能是由于所使用的体外检测的缺陷。为了系统地研究非同义ABCC2变体的功能后果，我们使用了一种新的筛选和插入(SCIN)技术，在HT1080细胞中实现了13个ABCC2变体的稳定和高重复性表达。Western blotting显示D333G、R1174H和R1181L的ABCC2表达低于野生型(100%)，而连锁突变体V1188E/C1515Y的ABCC2表达高于野生型(150%)。R1174H导致ABCC2错误定位于细胞质，呈内质网样分布。与WT相比，突变体N1244K和R1174H分别使GSMF和GSMCB的转运减少80%和50%，而R1181L和P1291L仅使GSMCB的转运减少50%。与蛋白质数据相反，双变异体V1188E/C1515Y使GSMF和GSMCB的特异性转运活性降低了40%。SCIN方法是系统分析ABCC2变异体功能特征的可行而可靠的方法。D333G、R1174H、R1181L、N1244K、P1291L和双变异体V1188E/C1515Y被认为是最有希望进行进一步临床评估的基因。哼唱，2012年33：750762。(C)2012年威利期刊公司。
The multidrug resistance-associated protein 2 (MRP2/ABCC2) is involved in the efflux of endogenous and xenobiotic substrates, including several anticancer and antiviral drugs. The functional consequences of ABCC2 protein variants remain inconsistent, which may be due to shortcomings of the in vitro assays used. To study systematically the functional consequences of nonsynonymous ABCC2 variants, we used a novel Screen and Insert (ScIn) technology to achieve stable and highly reproducible expression of 13 ABCC2 variants in HT1080 cells. Western blotting revealed lower (3065%) ABCC2 expression for D333G, R1174H, and R1181L as compared with wild type (WT; 100%), whereas the linked variant V1188E/C1515Y resulted in higher expression (150%). R1174H caused mislocalization of ABCC2 to the cytoplasm with an endoplasmic reticulum-like distribution. Variants N1244K and R1174H decreased transport of glutathionemethylfluorescein (GSMF) and glutathionemonochlorobimane (GSMCB) by 80% and 50%, respectively, whereas R1181L and P1291L reduced only GSMCB transport by 50% as compared with WT. Contrary to protein data, the double variant V1188E/C1515Y decreased specific transport activity for GSMF and GSMCB by 40%. The ScIn approach is a feasible and reliable method to functionally characterize systematically ABCC2 variants. D333G, R1174H, R1181L, N1244K, P1291L, and double variant V1188E/C1515Y have been identified as most promising for further clinical evaluation. Hum Mutat 33:750762, 2012. (c) 2012 Wiley Periodicals, Inc.